Prevention of hyperpigmentation (i.e. sun-burn) and aging is the key means for having healthy skins. However, many of the skin pigmentation and aging processes are associated with personal gene activities. For example, tyrosinase (Tyr), a melanocytic membrane-bound glycoprotein, is the rate-limiting enzyme critical for melanin (black pigment) biosynthesis in skins and hairs, while hyaluronidase (Hyal) often causes skin wrinkle by degrading subcutaneous hyaluronan (HA), the major anti-aging extracellular matrix in skins. Therefore, a good skin care can be achieved by suppressing these unwanted gene activities.
Currently, there is no prior art related to hyaluronidase inhibitor for wrinkle removal. For skin whitening and lightening, many prior arts attempting to inhibit tyrosinase function often use hormone-derived inhibitory peptides, small molecular chemicals and some plant extracts, including oligopeptides (e.g. U.S. Pat. No. 7,268,108 to Pinel; U.S. Pat. No. 6,852,699 to Schonrock), hydroxytetronic acid derivatives (e.g. U.S. Pat. No. 7,019,029 to Perricone), benzoyl compounds (e.g. U.S. Pat. No. 6,838,481 to Kim), hydroquinone compositions (e.g. U.S. Pat. Nos. 6,998,130 and 7,025,977 to Wortzman), alcohol diol and triol analogues (e.g. U.S. Pat. No. 7,250,157 to Brown), kojic acid derivatives (e.g. U.S. Pat. No. 6,710,076 to Ancira), ascomycete-derived enzymes (e.g. U.S. Pat. No. 6,514,506 to Mammone), and plant extracts (e.g. U.S. Pat. No. 7,192,617 to Nagamine; U.S. Pat. No. 7,125,572 to Lee; U.S. Pat. No. 6,521,267 to Steck; U.S. Pat. No. 7,105,184 to Pauly; U.S. Pat. Nos. 6,994,874, 7,060,304, and 7,247,321 to Leverett; U.S. Pat. Nos. 7,025,957, 7,029,709, and 7,097,866 to Arquette; U.S. Pat. Nos. 6,649,150 and 6,969,509 to Chaudhuri). Although these materials and methods may work well in vitro, only a few of them, such as hydroquinone and its derivatives, are able to induce good hypopigmenting effects in clinical trials (Solano et.al. (2006) Pigment Cell Res. 19: 550-571). Nevertheless, all hydroquinone derivatives leading to a reactive quinone are putative cytotoxic agents. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their safety and efficacy. (These publications and all other cited publications and patents in this application are hereby incorporated by reference as if fully set forth herein.)
With the advance of recent RNA interference (RNAi) technologies, novel small RNA agents have been found to provide more potent effects in targeted gene suppression, including the utilization of double-stranded short interfering RNA (e.g. dsRNA/siRNA) (Fire et al. (1998) Nature 391: 806-811; Elbashir et al. (2001) Nature 411: 494-498) and doxyribonucleotidylated-RNA interfering molecules (e.g. D-RNAi) (Lin et al. (2001) Biochem. Biophys. Res. Commun. 281: 639-644). Conceivably, these small RNA agents may be used to develop new cosmetic designs and products for skin care. In principle, the RNAi mechanism elicits a post-transcriptional gene silencing (PTGS) phenomenon capable of inhibiting specific gene function with high potency at a few nanomolar dosage, which has been proven to have lasting effect and much less toxic than conventional gene-knockout methods using antisense oligonucleotides or small molecule chemical inhibitors (Lin et al. (2001) Current Cancer Drug Targets 1: 241-247). As reported in many previous studies (Grant, S. R. (1999) Cell 96: 303-306; Elbashir et al. (2001) supra; Lin et al. (2001) supra; Lin et al. (2004a) Drug Design Reviews 1: 247-255), the siRNA-induced gene silencing effects may last over one week, while the D-RNAi effects can even sustain up to one month after one treatment. The siRNA/D-RNAi agents evoke a series of intracellular sequence-specific mRNA degradation and/or translational suppression processes, affecting all highly homologous gene transcripts, namely co-suppression. It has been observed that such co-suppression results from the generation of small RNA products (21-25 nucleotide bases) by the enzymatic activities of RNaseIII endoribonucleases (Dicer) and/or RNA-directed RNA polymerases (RdRp) on aberrant RNA templates, which are usually the derivatives of foreign transgenes or viral genomes (Grant, S. R. (1999) supra; Elbashir et al. (2001) supra; Lin et al. (2001) supra). Based on this well-established RNAi mechanism, prior arts attempting to inhibit tyrosinase function using synthetic siRNA and/or dsRNA agents include U.S. Pat. Application Publication No. 20050137151 to Binetti and U.S. Pat. Application Publication No. 20070134188 to Collin-Djangone.
Although the modern RNAi technologies may offer a new avenue for suppressing unwanted gene function in skins, the applications thereof have not been demonstrated to work constantly and safely in higher vertebrates, including fish, avian, mammal and human. For example, almost all of the siRNA agents are based on a double-stranded RNA (dsRNA) conformation, which has been shown to cause interferon-mediated non-specific RNA degradation in vertebrates (Stark et al. (1998) Annu. Rev. Biochem. 67: 227-264; Elbashir et al. supra; U.S. Pat. No. 4,289,850 to Robinson; U.S. Pat. No. 6,159,714 to Lau). Such an interferon-mediated cytotoxic response reduces the target specificity of siRNA-induced gene silencing effects and often results in global RNA degradation in vertebrate cells (Stark et.al. supra; Elbashir et al. supra). Especially in mammalian cells, it has been noted that the RNAi effects are disturbed when the siRNA/dsRNA size is longer than 25 base-pairs (bp) (Elbashir et al. supra). Transfection of siRNA or small hairpin RNA (shRNA) sized less than 25 bp may not completely overcome such a problem, because both Sledz et al. ((2003) Nat Cell Biol. 5: 834-839) and Lin et al. ((2004b) Intrn'l J Oncol. 24: 81-88) have reported that the high dosage of siRNAs and shRNAs (such as >250 nM in human T cells) is able to cause strong cytotoxic effects similar to those of long dsRNAs. This toxicity is due to their double-stranded RNA conformation, which activates the interferon-mediated non-specific RNA degradation and programmed cell death through the signaling pathways of cellular PKR and 2-5A systems. It is well known that interferon-induced protein kinase PKR can trigger cell apoptosis, while activation of interferon-induced 2′,5′-oligoadenylate synthetase (2-5A) system leads to extensive cleavage of single-stranded RNAs (i.e. mRNAs) (Stark et al. supra). Both PKR and 2-5A systems contain dsRNA-binding motifs, which possess high affinity to the double-stranded RNA conformation. Further, the most difficult problem is that it is impossible to deliver these small and unstable siRNA/shRNA constructs in vivo due to the abundant RNase activities in higher vertebrates (Brantl S. (2002) Biochimica et Biophysica Acta 1575, 15-25).
As the RNAi effects are naturally caused by the production of small RNA products (21-25 nucleotide bases) from a transcriptional template derived from foreign transgenes or viral genomes (Grant, S. R. (1999) supra; Lin et al. (2001) supra), recent utilization of Pol-III-mediated siRNA/shRNA expression vectors has offered relatively stable RNAi efficacy in vivo (Tuschl et al. (2002) Nat Biotechnol. 20: 446-448). Although prior arts (Miyagishi et al. (2002) Nat Biotechnol 20: 497-500; Lee et al. (2002) Nat Biotechnol 20: 500-505; Paul et al. (2002) Nat Biotechnol 20: 505-508) attempting to use such a vector-based siRNA approach have succeeded in maintaining constant gene silencing effects, their strategies failed to focus the RNAi effects on a targeted cell or tissue population because of the use of ubiquitous type III RNA polymerase (Pol-III) promoters. Pol-III promoters, such as U6 and H1, are activated in almost all cell types, making tissue-specific gene targeting impossible. Moreover, because the leaky read-through activity of Pol-III transcription often occurs on a short DNA template in the absence of proper termination, large RNA products longer than desired 25 bp can be synthesized and cause unexpected interferon cytotoxicity (Gunnery et al. (1999) J Mol Biol. 286: 745 757; Schramm et al. (2002) Genes Dev 16: 2593-2620). Such a problem can also result from the competitive conflict between the Pol-III promoter and another vector promoter (i.e. LTR and CMV promoters). Furthermore, it is recently noted that high siRNA/shRNA concentrations generated by the Pol-III-directed RNAi systems can over-saturate the cellular native microRNA (miRNA) pathway and thus cause global miRNA inhibition and cell death (Grimm et al. (2006) Nature 441: 537-541). These disadvantages discourage the use of Pol-III-based RNAi vector systems in health care.
In sum, in order to improve the delivery stability, targeting specificity and safety aspects of modern RNAi technologies for skin health care, a better induction and maintenance strategy is highly desired. Therefore, there remains a need for an effective, stable and safe gene modulation method as well as agent composition for suppressing unwanted gene function in skins, using the novel RNAi mechanisms.